Hybrid maize plant and seed 36D14

ABSTRACT

According to the invention, there is provided a hybrid maize plant, designated as 36D14, produced by crossing two Pioneer Hi-Bred International, Inc. proprietary inbred maize lines. This invention relates to the hybrid seed 36D14, the hybrid plant produced from the seed, and variants, mutants, and trivial modifications of hybrid 36D14. This invention also relates to methods for producing a maize plant containing in its genetic material one or more transgenes and to the transgenic maize plants produced by that method. This invention further relates to methods for producing maize lines derived from hybrid maize line 36D14 and to the maize lines derived by the use of those methods.

FIELD OF THE INVENTION

This invention is in the field of maize breeding, specifically relatingto hybrid maize designated 36D14.

BACKGROUND OF THE INVENTION Plant Breeding

Field crops are bred through techniques that take advantage of theplant's method of pollination. A plant is self-pollinated if pollen fromone flower is transferred to the same or another flower of the sameplant. A plant is cross-pollinated if the pollen comes from a flower ona different plant.

Plants that have been self-pollinated and selected for type for manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny. A cross between twodifferent homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Maize (Zea mays L.), often referred to as corn in the United States, canbe bred by both self-pollination and cross-pollination techniques. Maizehas separate male and female flowers on the same plant, located on thetassel and the ear, respectively. Natural pollination occurs in maizewhen wind blows pollen from the tassels to the silks that protrude fromthe tops of the ears.

The development of a hybrid maize variety in a maize plant breedingprogram involves three steps: (1) the selection of plants from variousgermplasm pools for initial breeding crosses; (2) the selfing of theselected plants from the breeding crosses for several generations toproduce a series of inbred lines, which, although different from eachother, breed true and are highly uniform; and (3) crossing the selectedinbred lines with unrelated inbred lines to produce the hybrid progeny(F1). During the inbreeding process in maize, the vigor of the linesdecreases. Vigor is restored when two different inbred lines are crossedto produce the hybrid progeny (F1). An important consequence of thehomozygosity and homogeneity of the inbred lines is that the hybridcreated by crossing a defined pair of inbreds will always be the same.Once the inbreds that create a superior hybrid have been identified, acontinual supply of the hybrid seed can be produced using these inbredparents and the hybrid corn plants can then be generated from thishybrid seed supply.

Large scale commercial maize hybrid production, as it is practicedtoday, requires the use of some form of male sterility system whichcontrols or inactivates male fertility. A reliable method of controllingmale fertility in plants also offers the opportunity for improved plantbreeding. This is especially true for development of maize hybrids,which relies upon some sort of male sterility system. There are severaloptions for controlling male fertility available to breeders, such as:manual or mechanical emasculation (or detasseling), cytoplasmic malesterility, genetic male sterility, gametocides and the like.

Hybrid maize seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twoinbred varieties of maize are planted in a field, and the pollen-bearingtassels are removed from one of the inbred (female) prior to pollenshed. Providing that there is sufficient isolation from sources offoreign maize pollen, the ears of the detasseled inbred will befertilized only from the other inbred (male), and the resulting seed istherefore hybrid and will form hybrid plants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent inmaize plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Usually seedfrom detasseled fertile maize and CMS produced seed of the same hybridare blended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., of Pioneer Hi-Bred, U.S. Patentapplication Ser. No. 5,432,068, have developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility; silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran antisense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see:Fabinjanski, et al. EPO 89/3010153.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, Glenn R., U.S. Pat. No.: 4,936,904). Applicationof the gametocide, timing of the application and genotype specificityoften limit the usefulness of the approach.

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. The development of maize hybrids in a maize plantbreeding program requires, in general, the development of homozygousinbred lines, the crossing of these lines, and the evaluation of thecrosses. Maize plant breeding programs combine the genetic backgroundsfrom two or more inbred lines or various other broad-based sources intobreeding pools from which new inbred lines are developed by selfing andselection of desired phenotypes. Hybrids also can be used as a source ofplant breeding material or as source populations from which to developor derive new maize lines. Plant breeding techniques known in the artand used in a maize plant breeding program include, but are not limitedto, recurrent selection backcrossing, pedigree breeding, restrictionlength polymorphism enhanced selection, genetic marker enhancedselection and transformation. The inbred lines derived from hybrids canbe developed using said methods of breeding such as pedigree breedingand recurrent selection. New inbreds are crossed with other inbred linesand the hybrids from these crosses are evaluated to determine which ofthose have commercial potential.

Recurrent selection breeding, backcrossing for example, can be used toimprove inbred lines and a hybrid which is made using those inbreds.Backcrossing can be used to transfer a specific desirable trait from oneinbred or source to an inbred that lacks that trait. This can beaccomplished, for example, by first crossing a superior inbred(recurrent parent) to a donor inbred (non-recurrent parent), thatcarries the appropriate gene(s) for the trait in question. The progenyof this cross is then mated back to the superior recurrent parentfollowed by selection in the resultant progeny for the desired trait tobe transferred from the non-recurrent parent. After five or morebackcross generations with selection for the desired trait and for thegermplasm inherited from the recurrent parent, the progeny will behomozygous for loci controlling the characteristic being transferred,but will be like the superior parent for essentially all other genes.The last backcross generation is then selfed to give pure breedingprogeny for the gene(s) being transferred. A hybrid developed frominbreds containing the transferred gene(s) is essentially the same as ahybrid developed from the same inbreds without the transferred gene(s).

There are many important factors to be considered in the art of plantbreeding, such as the ability to recognize important morphological andphysiological characteristics, the ability to design evaluationtechniques for genotypic and phenotypic traits of interest, and theability to search out and exploit the genes for the desired traits innew or improved combinations.

The objective of commercial maize hybrid line development resulting froma maize plant breeding program is to develop new inbred lines to producehybrids that combine to produce high grain yields and superior agronomicperformance. The primary trait breeders seek is yield. However, manyother major agronomic traits are of importance in hybrid combination andhave an impact on yield or otherwise provide superior performance inhybrid combinations. Such traits include percent grain moisture atharvest, relative maturity, resistance to stalk breakage, resistance toroot lodging, grain quality, and disease and insect resistance. Inaddition, the lines per se must have acceptable performance for parentaltraits such as seed yields, kernel sizes, pollen production, all ofwhich affect ability to provide parental lines in sufficient quantityand quality for hybridization. These traits have been shown to be undergenetic control and many if not all of the traits are affected bymultiple genes.

Pedigree Breeding

The pedigree method of breeding is the mostly widely used methodologyfor new hybrid line development.

In general terms this procedure consists of crossing two inbred lines toproduce the non-segregating F1 generation, and self pollination of theF1 generation to produce the F2 generation that segregates for allfactors for which the inbred parents differ. An example of this processis set forth below. Variations of this generalized pedigree method areused, but all these variations produce a segregating generation whichcontains a range of variation for the traits of interest.

Example 1 Hypothetical Example of Pedigree Breeding Program

Consider a cross between two inbred lines that differ for alleles at sixloci. The parental genotypes are:

Parent 1 A b C d e F/A b C d e F Parent 2 a B c D E f/a B c D E f

the F1 from a cross between these two parents is:

F1 A b C d e F/a B c D E f

Selfing F1 will produce an F2 generation including the followinggenotypes:

A B c D E f/a b C d e F  A B c D e f/a b C d E F A B c D e f/ a b C d eF . . .

The number of genotypes in the F2 is 36 for six segregating loci (729)and will produce (26)-2 possible new inbreds, (62 for six segregatingloci).

Each inbred parent which is used in breeding crosses represents a uniquecombination of genes, and the combined effects of the genes define theperformance of the inbred and its performance in hybrid combination.There is published evidence (Smith, O. S., J. S. C. Smith, S. L. Bowen,R. A. Tenborg and S. J. Wall, TAG 80:833-840 (1990)) that each of thelines are different and can be uniquely identified on the basis ofgenetically-controlled molecular markers.

It has been shown (Hallauer, Arnel R. and Miranda, J. B. Fo.Quantitative Genetics in Maize Breeding, Iowa State University Press,Ames Iowa, 1981) that most traits of economic value in maize are underthe genetic control of multiple genetic loci, and that there are a largenumber of unique combinations of these genes present in elite maizegermplasm. If not, genetic progress using elite inbred lines would nolonger be possible. Studies by Duvick and Russell (Duvick, D. N.,Maydica 37:69-79, (1992); Russell, W. A., Maydica XXIX:375-390 (1983))have shown that over the last 50 years the rate of genetic progress incommercial hybrids has been between one and two percent per year.

The number of genes affecting the trait of primary economic importancein maize, grain yield, has been estimated to be in the range of 10-1000.Inbred lines which are used as parents for breeding crosses differ inthe number and combination of these genes. These factors make the plantbreeder's task more difficult. Compounding this is evidence that no oneline contains the favorable allele at all loci, and that differentalleles have different economic values depending on the geneticbackground and field environment in which the hybrid is grown. Fiftyyears of breeding experience suggests that there are many genesaffecting grain yield and each of these has a relatively small effect onthis trait. The effects are small compared to breeders' ability tomeasure grain yield differences in evaluation trials. Therefore, theparents of the breeding cross must differ at several of these loci sothat the genetic differences in the progeny will be large enough thatbreeders can develop a line that increases the economic worth of itshybrids over that of hybrids made with either parent.

If the number of loci segregating in a cross between two inbred lines isn, the number of unique genotypes in the F2 generation is 3n and thenumber of unique inbred lines from this cross is {(2n)−2}. Only a verylimited number of these combinations are useful. Only about 1 in 10,000of the progeny from F2's are commercially useful.

By way of example, if it is assumed that the number of segregating lociin F2 is somewhere between 20 and 50, and that each parent is fixed forhalf the favorable alleles, it is then possible to calculate theapproximate probabilities of finding an inbred that has the favorableallele at {(n/2)+m} loci, where n/2 is the number of favorable allelesin each of the parents and m is the number of additional favorablealleles in the new inbred. See Example 2 below. The number m is assumedto be greater than three because each allele has so small an effect thatevaluation techniques are not sensitive enough to detect differences dueto three or less favorable alleles. The probabilities in Example 2 areon the order of 10-5 or smaller and they are the probabilities that atleast one genotype with (n/2)=m favorable alleles will exist.

To put this in perspective, the number of plants grown on 60 millionacres (approximate United States corn acreage) at 25,000 plants/acre is1.5×1012.

Example 2

Probability of finding an inbred with m of n favorable alleles. Assumeeach parent has n/2 of the favorable alleles and only ½ of thecombinations of loci are economically useful. No. of No of favorable Noadditional segregating alleles in Parents favorable alleles Probabilitythat loci (n) (n/2) in new inbred genotype occurs* 20 10 14 3 × 10⁻⁵ 2412 16 2 × 10⁻⁵ 28 14 18 1 × 10⁻⁵ 32 16 20 8 × 10⁻⁶ 36 18 22 5 × 10⁻⁶ 4020 24 3 × 10⁻⁶ 44 22 26 2 × 10⁻⁶ 48 24 28 1 × 10⁻⁶ *Probability that auseful combination exists, does not include the probability ofidentifying this combination if it does exist.

The possibility of having a usably high probability of being able toidentify this genotype based on replicated field testing would be mostlikely smaller than this, and is a function of how large a population ofgenotypes is tested and how testing resources are allocated in thetesting program.

SUMMARY OF THE INVENTION

According to the invention, there is provided a hybrid maize plant,designated as 36D14, produced by crossing two Pioneer Hi-BredInternational, Inc. proprietary inbred maize line GE518397 and GE501400.These lines, deposited with the American Type Culture Collection,(ATCC), Manassas, Va. 20110, have ATCC Deposit No. PTA-1728 forGE518397, which was deposited with the ATCC on Apr. 18, 2000, and ATCCDeposit No. PTA-1282 for GE501400, which was deposited with the ATCC onFeb. 3, 2000. The seeds deposited with the ATCC were taken from thedeposit maintained by Pioneer Hi-Bred International, Inc., 800 CapitalSquare, 400 Locust Street, Des Moines, Iowa 50309-2340 since prior tothe filing date of this application. This invention thus relates to thehybrid seed 36D14, the hybrid plant produced from the seed, andvariants, mutants and trivial modifications of hybrid 36D14. Thisinvention also relates to methods for producing a maize plant containingin its genetic material one or more transgenes and to the transgenicmaize plants produced by that method. This invention further relates tomethods for producing maize lines derived from hybrid maize line 36D14and to the maize lines derived by the use of those methods. This hybridmaize plant is characterized by consistent grain yield and above averagelate season plant health. Hybrid 36D14 displays a substantial degree ofimidazolinone resistance.

DEFINITIONS

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. NOTE: ABS is in absolute termsand % MN is percent of the mean for the experiments in which the inbredor hybrid was grown. These designators will follow the descriptors todenote how the values are to be interpreted. Below are the descriptorsused in the data tables included herein.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance.

BAR PLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BRT STK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap in pairedcomparisons and on a 1 to 9 scale (9=highest resistance) inCharacteristics Charts.

BU ACR=YIELD (BUSHELS/ACRE). Yield of the grain at harvest in bushelsper acre adjusted to 15.5% moisture.

CLN=CORN LETHAL NECROSIS (synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV)). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance.

COM RST=COMMON RUST (Puccinia sorghi). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance.

CRM=COMPARATIVE RELATIVE MATURITY (see PRM).

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1-9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

D/E=DROPPED EARS. Represented in a 1 to 9 scale in the CharacteristicsChart, where 9 is the rating representing the least, or no, droppedears.

DIP ERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance.

DRP EAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest.

D/T=DROUGHT TOLERANCE. This represents a 1-9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance.

EAR HT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured in inches.This is represented in a 1 to 9 scale in the Characteristics Chart,where 9 is highest.

EAR MLD=General Ear Mold. Visual rating (1-9 score) where a “1” is verysusceptible and a “9” is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold.

EAR SZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

ECB 1LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance.

ECB2IT=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilalis). Average inches of tunneling per plant in thestalk.

ECB 2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by European Corn Borer, Second Generation.A higher scone indicates a higher resistance.

ECB DPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation corn borer infestation.

E/G=EARLY GROWTH. This represents a 1 to 9 rating for early growth,scored when two leaf collars are visible.

EST CNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYE SPT=Eye Spot (Kabatiella zeae or Aureobasidium zeae). A 1 to 9visual rating indicating the resistance to Eye Spot. A higher scoreindicates a higher resistance.

FUS ERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium ear rot. A higher score indicates a higher resistance.

G/A=GRAIN APPEARANCE. Appearance of grain in the grain tank (scored downfor mold, cracks, red streak, etc.).

GDU=Growing Degree Units. Using the Barger Heat Unit Theory, thatassumes that maize growth occurs in the temperature range 50° F.-86° F.and that temperatures outside this range slow down growth; the maximumdaily heat unit accumulation is 36 and the minimum daily heat unitaccumulation is 0. The seasonal accumulation of GDU is a major factor indetermining maturity zones.

GDU PHY=GDU TO PHYSIOLOGICAL MATURITY. The number of growing degreeunits required for an inbred or hybrid line to have approximately 50percent of plants at physiological maturity from time of planting.Growing degree units are calculated by the Barger method.

GDU SHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated be Barer Method, where theheat units for a 24-hour period are:${GDU} = {\frac{\left( {{Max}.\quad {temp}.{+ \quad {{Min}.\quad {temp}.}}} \right)}{2} - 50}$

The highest maximum temperature used is 86° F. and the lowest minimumtemperature used is 50° F. For each inbred or hybrid it takes a certainnumber of GDUs to reach various stages of plant development.

GDU SLK=GDU TO SILK. The number of growing degree units required for allinbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GIB ERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae). A 1 to 9visual rating indicating the resistance to Gibberella Ear Rot. A higherscore indicates a higher resistance.

GLF SPT=Gray Leaf Spot (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance.

GOS WLT=Goss' Wilt (Corynebacterium nebraskense). A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance.

GRN APP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain quality.

H/POP=YIELD AT HIGH DENSITY. Yield ability at relatively high plantdensities on 1-9 relative rating system with a higher number indicatingthe hybrid responds well to high plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to increased plant density.

HC BLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporiumcarbonum). A 1 to 9 visual rating indicating the resistance toHelminthosporium infection. A higher score indicates a higherresistance.

HD SMT=Head Smut (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridor per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. GROSS INCOME advantage of variety #1over variety #2.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1-9 relative system with a higher number indicating thehybrid responds well to low plant densities for yield relative to otherhybrids. A 1, 5, and 9 would represent very poor, average, and very goodyield response, respectively, to low plant density.

MDM CPX=Maize Dwarf Mosaic Complex (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance.

MST=HARVEST MOISTURE. The moisture is the actual percentage moisture ofthe grain at harvest.

MST ADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2 —MOISTURE of variety#1 MOISTURE ADVANTAGE of variety #1.

NLF BLT=Northern Leaf Blight (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance.

OIL=GRAIN OIL. The amount of the kernel that is oil, expressed as apercentage on a dry weight basis.

PHY CRM=CRM at physiological maturity.

PLT HT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in inches. This is represented as a1 to 9 scale, 9 highest, in the Characteristics Chart.

POL SC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POL WT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

POP K/A=PLANT POPULATIONS. Measured as 1000s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2 —PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED Relative Maturity. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRM SHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PRO=PROTEIN RATING. Rating on a 1 to 9 scale comparing relative amountof protein in the grain compared to hybrids of similar maturity. A “1”score difference represents a 0.4 point change in grain protein percent(e.g., 8.0% to 8.4%).

PROTEIN=GRAIN PROTEIN. The amount of the kernel that is crude protein,expressed as a percentage on a dry weight basis.

P/Y=PROTEIN YIELD RATING. Indicates, on a 1 to 9 scale, the economicvalue of a hybrid for swine and poultry feeders. This takes into accountthe income due to yield moisture and protein content.

ROOTS (%)=Percent of stalks NOT root lodged at harvest.

R/L=ROOT LODGING. A 1 to 9 rating indicating the level of root lodgingresistance. The higher score represents higher levels of resistance.

RT LDG=ROOT LODGING. Root lodging is the percentage of plants that donot root lodge; plants that lean from the vertical axis as anapproximately 30° angle or greater would be counted as root lodged.

RTL ADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2.

S/L=STALK LODGING. A 1 to 9 rating indicating the level of stalk lodgingresistance. The higher scores represent higher levels of resistance.

SCT GRN=SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter grain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SDG VGR=SEEDLING VIGOR. This is the visual rating (1 to 9) of the amountof vegetative growth after emergence at the seedling stage(approximately five leaves). A higher score indicates better vigor.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SLF BLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance.

SLK CRM=CRM at Silking.

SOU RST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance.

STA GRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STAND (%)=Percent of stalks standing at harvest.

STARCH=GRAIN STARCH. The amount of the kernel that is starch, expressedas a percentage on a dry weight basis.

STD ADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STK CNT=NUMBER OF PLANTS. This is the final stand or number of plantsper plot.

STK LDG=STALK LODGING. This is the percentage of plants that did notstalk lodge (stalk breakage) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear.

STW WLT=Stewart's Wilt (Erwinia stewartii). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance.

TAS BLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting.

TAS SZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TEX EAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TIL LER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TST WT (CHARACTERISTICS CHART)=Test weight on a 1 to 9 rating scale witha 9 being the highest rating.

TST WT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grainin pounds for a given volume (bushel).

TST WTA=TEST WEIGHT ADJUSTED. The measure of the weight of the grain inpounds for a given volume (bushel) adjusted for 15.5 percent moisture.

TSW ADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M %=PERCENT MOISTURE WINS.

WIN Y %=PERCENT YIELD WINS.

YLD=YIELD. It is the same as BU ACR ABS.

YLD ADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1—YIELD variety #2 yieldadvantage of variety #1.

YLD SC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

DETAILED DESCRIPTION OF THE INVENTION

Pioneer Brand Hybrid 36D14 has consistent grain yield. The hybrid showsabove average late season plant health and above average stalk lodgingresistance. 36D14 further exhibits above average tolerance to NorthernCorn Leaf Blight, Stewart's Wilt, and Anthracnose Stalk Rot. 36D14 alsoshows above average brittle snap resistance and improved resistance toGray Leaf Spot. 36D14 demonstrates a substantial degree of imidazolinoneresistance. It is particularly suited to the Central Corn Belt and theNorthwest, North Central, Northeast, and Western regions of the UnitedStates and also to Canada, including Eastern Ontario.

Pioneer Brand Hybrid 36D14 is a single cross, yellow endosperm, dentmaize hybrid. Hybrid 36D14 has a relative maturity of approximately 103based on the Comparative Relative Maturity Rating System for harvestmoisture of grain.

This hybrid has the following characteristics based on the datacollected primarily at Johnston, Iowa.

TABLE 1 VARIETY DESCRIPTION INFORMATION VARIETY = 36D14 1. TYPE:(describe intermediate types in Comments section): 2 1 = Sweet 2 = Dent3 = Flint 4 = Flour 5 = Pop 6 = Ornamental 2. MATURITY: HEAT DAYS UNITS064 1,226.7 From emergence to 50% of plants in silk 065 1,252.7 Fromemergence to 50% of plants in pollen 002 0,050.7 From 10% to 90% pollenshed 079 1,724.0 From 50% silk to harvest at 25% moisture StandardSample Deviation Size 3. PLANT: 0,282.7 cm Plant Height (to tassel tip)24.01 15 0,103.3 cm Ear Height (to base of top ear node) 21.39 150,019.2 cm Length of Top Ear Internode 2.31 15 0.1 Average Number ofTillers 0.13  3 1.0 Average Number of Ears per Stalk 0.00  3 2.0Anthocyanin of Brace Roots: 1 = Absent 2 = Faint 3 = Moderate 4 = Dark4. LEAF: 009.7 cm Width of Ear Node Leaf 0.46 15 088.0 cm Length of EarNode Leaf 6.50 15 05.7 Number of leaves above top ear 0.61 15 042.0Degrees Leaf Angle (measure from 2nd leaf 5.00 15 above ear at anthesisto stalk above leaf) 03 Leaf Color  Dark Green (Munsell code) 5GY36 1.0Leaf Sheath Pubescence (Rate on scale from 1 = none to 9 = like peachfuzz) 6.3 Marginal Waves (Rate on scale from 1 = none to 9 = many) 6.0Longitudinal Creases (Rate on scale from 1 = none to 9 = many) 5.TASSEL: 08.3 Number of Primary Lateral Branches 2.80 15 029.3 BranchAngle from Central Spike 15.28 15 61.7 cm Tassel Length (from top leafcollar to tassel tip) 5.85 15 6.7 Pollen Shed (rate on scale from 0 =male sterile to 9 = heavy shed) 11 Anther Color  Pink (Munsell code)10RP58 01 Glume Color  Light Green (Munsell code) 5GY76 1.0 Bar Glumes(Glume Bands): 1 = Absent 2 = Present 6a. EAR (Unhusked Data): 14 SilkColor (3 days after emergence) Red (Munsell code) 2.5R48 1 Fresh HuskColor (25 days after 50% silking) Light Green (Munsell code) 5GY68 21Dry Husk Color (65 days after 50% silking) Buff (Munsell code) 5Y92 1Position of Ear at Dry Husk Stage: 1 = Upright Upright 2 = Horizontal 3= Pendant 7 Husk Tightness (Rate of Scale from 1 = very loose to 9 =very tight) 2 Husk Extension (at harvest): 1 = Short (ears exposed) 2 =Medium (<8 cm) 3 = Long (8-10 cm beyond ear tip) 4 = Very Medium Long(>10 cm) 6b. EAR (Husked Ear Data) 19 cm Ear Length 0.58 15 44 mm EarDiameter at mid-point 1.53 15 210 gm Earweight 14.64 15 13 Number ofKernel Rows 1.15 15 2 Kernel Rows: 1 = Indistinct 2 = Distinct Distinct1 Row Alignment: 1 = Straight 2 = Slightly Straight Curved 3 = Spiral 10cm Shank Length 1.73 15 1 Ear Taper: 1 = Slight 2 = Average Slight 3 =Extreme 7. KERNEL (Dried): 12 mm Kernel Length 0.58 15 9 mm Kernel Width0.58 15 4 mm Kernel Thickness 0.00 15 21 % Round Kernels (Shape Grade)4.73  3 1 Aleurone Color Pattern: 1 = Homozygous 2 = HomozygousSegregating 7 Aluerone Color  Yellow (Munsell code) 10YR814 7 HardEndosperm Color  Yellow (Munsell code) 10YR714 3 Endosperm Type:  NormalStarch 1 = Sweet (Su1) 2 = Extra Sweet (sh2) 3 = Normal Starch 4 = HighAmylose Starch 5 = Waxy Starch 6 = High Protein 7 = High Lysine 8 =Super Sweet (se) 9 = High Oil 10 = Other_(—) 48 gm Weight per 100Kernels (unsized sample) 2.00  3 8. COB: 21 mm Cob Diameter at mid-point0.58 15 9. DISEASE RESISTANCE (Rate from 1 (most susceptible) to 9 (mostresistant): leave blank if not tested; leave Race or Strain Optionsblank if polygenic): A. Leaf Blights, Wilts, and Local InfectionDiseases Anthracnose Leaf Blight (Colletotrichum graminicola) CommonRust (Puccinia sorghi) Common Smut (Ustilago maydis) 6 Eyespot(Kabatiella zeae) Goss's Wilt (Clavibacter michiganense spp.nebraskense) 5 Gray Leaf Spot (Cercospora zeae-maydis) HelminthosporiumLeaf Spot (Bipolaris zeicola) Race_(—) 6 Northern Leaf Blight(Exserohilum turcicum) Race_(—) Southern Leaf Blight (Bipolaris maydis)Race_(—) Southern Rust (Puccinia polysora) 6 Stewart's Wilt (Erwiniastewartii) Other (Specify)_(—) B. Systemic Diseases Corn Lethal Necrosis(MCMV and MDMV) Head Smut (Sphacelotheca reiliana) Maize Chlorotic DwarfVirus (MDV) Maize Chlorotic Mottle Virus (MCMV) Maize Dwarf Mosaic Virus(MDMV) Sorghum Downy Mildew of Corn (Peronosclerospora sorghi) Other(Specify)_(—) C. Stalk Rots 5 Anthracnose Stalk Rot (Colletotrichumgraminicola) Diplodia Stalk Rot (Stenocarpella maydis) Fusarium StalkRot (Fusarium moniliforme) Gibberella Stalk Rot (Gibberella zeae) Other(Specify)_(—) D. Ear and Kernel Rots Aspergillus Ear and Kernel Rot(Aspergillus flavus) Diplodia Ear Rot (Stenocarpella maydis) 5 FusariumEar and Kernel Rot (Fusarium moniliforme) 8 Gibberella Ear Rot(Gibberella zeae) Other (Specify)_(—) 10. INSECT RESISTANCE (Rate from 1(most susceptible) to 9 (most resistant); (leave blank if not tested)Banks grass Mite (Oligonychus pratensis) Corn Worm (Helicoverpa zea) Leaf Feeding  Silk Feeding  mg larval wt. Ear Damage Corn Leaf Aphid(Rhopalosiphum maidis) Corn Sap Beetle (Carpophilus dimidiatus) EuropeanCorn Borer (Ostrinia nubilalis) 5  1st Generation (Typically Whorl LeafFeeding) 3  2nd Generation (Typically Leaf Sheath-Collar Feeding)  StalkTunneling cm tunneled/plant Fall Armyworm (Spodoptera fruqiperda)  LeafFeeding  Silk Feeding  mg larval wt. Maize Weevil (Sitophilus zeamaize)Northern Rootworm (Diabrotica barberi) Southern Rootworm (Diabroticaundecimpunctata) Southwestern Corn Borer (Diatreaea grandiosella)  LeafFeeding  Stalk Tunneling  cm tunneled/plant Two-Spotted Spider Mite(Tetranychus urticae) Western Rootworm (Diabrotica virgifrea virgifera)Other (Specify)_(—) 11. AGRONOMIC TRAITS: 6 Staygreen (at 65 days afteranthesis) (Rate on a scale from 1 = worst to 9 = excellent) 1.4 %Dropped Ears (at 65 days after anthesis) % Pre-anthesis Brittle Snapping% Pre-anthesis Root Lodging 20.8 Post-anthesis Root Lodging (at 65 daysafter anthesis) 11,111 Kg/ha Yield (at 12-13% grain moisture) *Ininterpreting the foregoing color designations, reference may be made tothe Munsell Glossy Book of Color, a standard color reference.

Research Comparisons for Pioneer Hybrid 36D14

Comparisons of characteristics for Pioneer Brand Hybrid 36D14 were madeagainst Pioneer Brand Hybrids 3751IR, 35A19, and 37J99, all of which aresimilar in area of adaptation to hybrid 36D14.

Table 2A compares Pioneer Brand Hybrid 36D14 and Pioneer Brand Hybrid3751IR. The results indicate that hybrid 36D14 has significantly higherharvest moisture but is significantly higher yielding than hybrid3751IR. The table has few repetitions of data from few locations, it isexpected that other significant differences will be observed withadditional data.

Table 2B compares Pioneer Brand Hybrid 36D14 and Pioneer Brand Hybrid35A19. As can be seen from the results hybrid 36D14 is significantlyhigher yielding with significantly lower test weight than hybrid 35A19.Hybrid 36D14 also demonstrates, significantly shorter plant stature andis earlier to flower with a significantly lower number of growing degreeunits to pollen shed and to silk than hybrid 35A19. Hybrid 36D14 furtherdemonstrates significantly superior resistance to brittle snap andsignificantly better tolerance to Stewart's Wilt than hybrid 35A1 9.

The results in Table 2C compare Pioneer Brand Hybrid 36D14 with PioneerBrand Hybrid 37J99. The table indicates that hybrid 36D14 issignificantly higher yielding but with significantly higher harvestmoisture than hybrid 37J99. 36D14 also exhibits a significantly superiorstay green score and is later to flower with a significantly highernumber of growing degree units to pollen shed and to silk than hybrid37J99. Hybrid 36D14 further demonstrates significantly lower earplacement and significantly better tolerance to Stewart's Wilt thanhybrid 37J99.

TABLE 2A HYBRID COMPARISON REPORT VARIETY #1 = 36D14 VARIETY #2 = 3751IRBU BU TST STK RT STK BRT ACR ACR MST WT CNT LDG LDG STK ABS % MN % MNABS % MN % HN % MN % MN TOTAL SUM 1 191.5 103 102 55.5 100 102 100 101 2176.0 95 95 54.3 99 115 102 102 LOCS 6 6 6 1 6 4 3 1 REPS 54 54 54 6 5436 24 12 DIFF 15.5 8 7 1.1 1 13 2 0 PR > T .026+ .019+ .000# .269 .641.686 *= 10% SIG += 5% SIG #= 1% SIG

TABLE 2B HYBRID COMPARISON REPORT VARIETY #1 = 36D14 VARIETY #2 = 35A19PRM BU BU TST SDG EST GDU GDU STK PLT PRM SHD ACR ACR MST WT VGR CNT SHDSLK CNT HT ABS ABS ABS % MN % MN ABS % MN % MN % MN % MN % MN % MN TOTALSUM 1 103 102 182.3 104 100 55.3 99 103 99 99 101 99 2 103 106 165.2 9499 56.8 106 101 104 104 101 107 LOCS 1 1 76 76 77 34 14 7 23 16 102 25REPS 1 1 163 163 164 66 14 7 24 16 205 36 DIFF 0 4 17.1 10 1 1.5 7 2 5 50 8 PR > T .000# .000# .145 .000# .240 .291 .000# .000# .999 .000# EARRT STA STK BRT DRP GLF NLF GOS STW ANT FUS GIB HT LDG GRN LDG STK EARSPT BLT WLT WLT ROT ERS ERS % MN % MN % MN % MN % MN % MN ABS ABS ABSABS ABS ABS ABS TOTAL SUM 1 97 93 109 102 102 100 5.0 5.7 6.0 5.8 5.15.0 7.5 2 96 108 112 99 91 100 4.7 4.7 7.0 3.6 5.5 4.0 6.8 LOCS 25 28 2029 30 6 5 3 1 5 4 1 2 REPS 36 50 20 40 47 6 8 5 2 5 6 1 3 DIFF 0 15 2 411 0 0.3 1.0 1.0 2.2 0.4 1.0 0.8 PR > T .999 .015+ .824 .365 .007# .999.607 .574 .029+ .650 .795 * = 10% SIG += 5% SIG #= 1% SIG

TABLE 2C HYBRID COMPARISON REPORT VARIETY #1 = 36D14 VARIETY #2 = 37J99PRM BU BU TST SDG EST GDU GDU STK PLT EAR PRM SHD ACR ACR MST WT VGR CNTSHD SLK CNT HT HT ABS ABS ABS % MN % MN ABS % MN % MN % MN % MN % MN %MN % MN TOTAL 1 103 102 184.1 104 101 55.3 99 103 99 99 101 99 97 SUM 2100 99 167.8 94 91 54.8 104 104 96 96 101 100 102 LOCS 1 1 74 74 75 3314 7 23 16 100 25 25 REPS 1 1 122 122 123 38 14 7 24 16 161 26 26 DIFF 43 16.3 10 10 0.4 6 1 3 3 0 1 5 PR > T .000# .000# .000# .102 .126 .615.000# .000# .999 .286 .029+ RT STA STK BRT DRP GLF NLF GOS STW ANT FUSGIB ECB ECB LDG GRN LDG STK EAR SPT BLT WLT WLT ROT ERS ERS 1LF 2SC % MN% MN % MN % MN % MN ABS ABS ABS ABS ABS ABS ABS ABS ABS TOTAL 1 94 109102 103 100 5.0 5.7 6.0 5.8 5.8 5.0 7.5 5.3 3.3 SUM 2 92 81 101 101 1004.4 4.5 5.5 3.4 5.0 7.0 7.8 4.8 2.8 LOCS 30 20 31 30 6 5 3 1 5 3 1 2 2 2REPS 60 20 51 43 6 8 5 2 5 5 1 3 4 4 DIFF 2 28 1 2 0 0.6 1.2 0.5 2.4 0.82.0 0.3 0.5 0.5 PR > T .524 .000# .274 .189 .999 .358 .118 .033+ .300.795 .500 .705 *= 10% SIG += 5% SIG #= 1% SIG

Strip Test Data for Hybrid 36D14

Comparison data was collected from strip tests that were grown byfarmers. Each hybrid was grown in strips of 4, 6, 8, 12, etc. rows infields depending on the size of the planter used. The data was collectedfrom strip tests that had the hybrids in the same area and weighed. Themoisture percentage was determined and bushels per acre was adjusted to15.5 percent moisture. The number of comparisons represent the number oflocations or replications for the two hybrids that were grown in thesame field in close proximity and compared.

Comparison strip testing was done between Pioneer Brand Hybrid 36D14 andPioneer Brand Hybrids 35A19, and 37J99. The comparisons come from allthe hybrid's adapted growing areas in the United States.

These results are presented in Table 3. As can be seen from the tablehybrid 36D14 demonstrates a yield advantage over each of the hybridscompared. The average yield advantage is 10.2 bushels per acre. Hybrid36D14 also demonstrates a greater average income advantage to the farmerbased on adjusted growth income over each of the comparison hybrids. Theaverage income advantage was $17.16 per acre. Hybrid 36D14's yield andincome advantage plus its advantage for other characteristics over thesehybrids will make it an important addition for most of the areas wherethese hybrids are grown.

TABLE 3 1998 PERFORMANCE COMPARISON REPORT FOR CORN 1 YEAR SUMMARY OFALL STANDARD TEST TYPES Income/ Pop Stand Roots Test Brand Product YieldMoist Acre K/Acre (%) (%) Wt Pioneer 36D14 179.6 18.9 344.31 27.0 93 8956.9 Pioneer 35A19 171.3 18.7 329.26 26.8 93 93 58.3 Advantage  8.3 −.2 15.05  .2  0 −4  −1.4 Number of Comparisons 120  120  120  99 61 50111  Percent Wins 69 42 68 36 34 16  6 Probability of Difference 99 7199 83 51 62 99 Pioneer 36D14 185.4 19.5 353.12 27.0 92 89 56.6 Pioneer37J99 171.8 18.1 332.25 26.7 90 82 56.0 Advantage  13.6  −1.4  20.87  .3 2  7  .6 Number of Comparisons 68 68 68 58 34 26 64 Percent Wins 91 1088 57 53 50 66 Probability of Difference 99 99 99 77 11 11 88 Pioneer36D14 181.7 19.1 347.50 27.0 93 89 56.8 Weighted Avg 171.5 18.5 330.3426.7 92 89 57.5 Advantage  10.2  −.6  17.16  .3  1  0  −.7 Number ofComparisons 188  188  188  157  95 76 175  Percent Wins 77 30 76 44 4128 28 Probability of Difference 99 99 99 92 27 42 71 NOTE: Theprobability values are useful in analyzing if there is a “real”difference in the genetic potential of the products involved. Highvalues are desirable, with 95% considered significant for realdifferences.

Comparison of Key Characteristics for Hybrid 36D14

Characteristics of Pioneer Hybrid 36D14 are compared to Pioneer Hybrids3751IR, 35A19, and 37J99 in Table 4. The values given for most traitsare on a 1-9 basis. In these cases 9 would be outstanding, while 1 wouldbe poor for the given characteristics. Table 4 shows that hybrid 36D14has excellent yield with a score of 9. Hybrid 36D14 also exhibits verygood tolerance to Gray Leaf Spot, Northern Leaf Blight, Stewart's Wiltand Anthracnose Stalk Rot with scores that are higher than any of thecomparison hybrids. Hybrid 36D14's excellent yield combined with itsother favorable agronomic characteristics should make it an importanthybrid to its area of adaptation.

TABLE 4 Hybrid Patent Comparisons - Characteristics Pioneer Hybrid 36D14vs. Pioneer Hybrids 3751IR, 35A19, 37J99 SILK PHY GDU GDU STA TSTVARIETY CRM CRM CRM SILK PHY YLD H/POP L/POP D/D S/L R/L GRN D/T WT E/G36D14 103 102 103 1270 2470 9 6 6 4 6 6 4 5 3751IR  97 100 100 1250 23908 8 8 7 4 4 5 7 4 4 35A19 104 106 106 1320 2550 8 8 7 6 6 7 7 7 8 437J99  97  98  99 1230 2370 9 9 8 7 4 4 6 7 4 6 PLT EAR BRT HSK GLF NLFGOS STW ANT HD FUS GIB EYE ECB ECB VARIETY HT HT D/E TK CV SPT BLT WLTWLT ROT SMT ERS ERS SPT 1LF 2SC 36D14 5 5 4 6 5 5 6 6 6 5 3 3751IR 5 4 56 5 3 5 5 8 6 4 6 5 3 35A19 8 4 7 4 5 4 5 8 4 5 6 4 5 7 3 6 37J99 6 6 54 6 4 5 6 4 5 7 5 5 4 3

Further Embodiments of the Invention

This invention includes hybrid maize seed of 36D14 and the hybrid maizeplant produced therefrom. The foregoing was set forth by way of exampleand is not intended to limit the scope of the invention.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants, orparts of plants, such as embryos, pollen, ovules, flowers, kernels,ears, cobs, leaves, seeds, husks, stalks, roots, root tips, anthers,silk and the like.

Duncan, Williams, Zehr, and Widholm, Planta, (1985) 165:322-332 reflectsthat 97% of the plants cultured which produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus which produced plants. In a furtherstudy in 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262-265 reports several media additions which enhance regenerabilityof callus of two inbred lines. Other published reports also indicatedthat “nontraditional” tissues are capable of producing somaticembryogenesis and plant regeneration. K. P. Rao, et al., Maize GeneticsCooperation Newsletter, 60:64-65 (1986), refers to somatic embryogenesisfrom glume callus cultures and B. V. Conger, et al., Plant Cell Reports,6:345-347 (1987) indicates somatic embryogenesis from the tissuecultures, of maize leaf segments. Thus, it is clear from the literaturethat the state of the art is such that these methods of obtaining plantsare, and were, “conventional” in the sense that they are routinely usedand have a very high rate of success.

Tissue culture of maize is described in European Patent Application,publication 160,390, incorporated herein by reference. Maize tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 1982, at367-372) and in Duncan, et al., “The Production of Callus Capable ofPlant Regeneration from Immature Embryos of Numerous Zea MaysGeneotypes,” 165 Planta 322-332 (1985). Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce maize plants having the genotype of 36D14.

Transformation of Maize

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native orendogenous genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreign,additional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transgenicversions of the claimed hybrid maize line 36D14.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used, alone or incombination with other plasmids, to provide transformed maize plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the maize plant(s).

Expression Vectors For Maize Transformation

Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e. inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80: 4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210: 86 (1987), Svab etal., Plant Mol.. Biol.. 14: 197 (1990), Hille et al., Plant Mol. Biol.7: 171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618(1990) and Stalker et al., Science 242: 419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67(1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant CellRep. 8: 643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343(1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984). Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway. Ludwig et al., Science 247: 449(1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes Publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115: 15la (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263: 802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissues arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inmaize. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in maize. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol.22: 361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al. PNAS 90: 4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243: 32-38 (1994)) or Tet repressor from Tn10 (Gatzet al., Mol. Gen. Genet. 227: 229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inmaize or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in maize.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313: 810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol 12: 619-632 (1989) andChristensen et al., Plant Mol. Biol. 18: 675-689 (1992)): pEMU (Last etal., Theor. Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBOJ. 3: 2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231: 276-285 (1992) and Atanassova et al., Plant Journal 2(3):291-300 (1992)).

The ALS promoter, a XbaI/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence that has substantial sequencesimilarity to said XbaI/NcoI fragment), represents a particularly usefulconstitutive promoter. See PCT application WO96/30530.

C. Tissue-specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin maize. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in maize. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,-such as that from the phaseolin gene (Murai et al., Science 23: 476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11): 2723-2729(1985) and Timko et al., Nature 318: 579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genet. 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genet.224: 161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6: 217-224 (1993).

Signal Sequences For Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized. The presence of asignal sequence directs a polypeptide to either an intracellularorganelle or subcellular compartment or for secretion to the apoplast.Many signal sequences are known in the art. See, for example, Becker etal., Plant Mol. Biol.20: 49 (1992), Close, P. S., Master's Thesis, IowaState University (1993), Knox, C., et al., “Structure and Organizationof Two Divergent Alpha-Amylase Genes From Barley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91: 124-129 (1989), Fontes etal., Plant Cell 3: 483-496 (1991), Matsuoka et al., Proc. Natl. Acad.Sci. 88: 834 (1991), Gould et al., J. Cell Biol 108: 1657 (1989),Creissen et al., Plant J. 2: 129 (1991), Kalderon, D., Robers, B.,Richardson, W., and Smith A., “A short amino acid sequence able tospecify nuclear location”, Cell 39: 499-509 (1984), Stiefel, V.,Ruiz-Avila, L., Raz R., Valles M., Gomez J., Pages M.,Martinez-Izquierdo J., Ludevid M., Landale J., Nelson T., andPuigdomenech P., “Expression of a maize cell wall hydroxyproline-richglycoprotein gene in early leaf and root vascular differentiation”,Plant Cell 2: 785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is maize. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, and Simple Sequence Repeats (SSR) which identifies theapproximate chromosomal location of the integrated DNA molecule. Forexemplary methodologies in this regard, see Glick and Thompson, METHODSIN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284 (CRC Press, BocaRaton, 1993). Map information concerning chromosomal location is usefulfor proprietary protection of a subject transgenic plant. Ifunauthorized propagation is undertaken and crosses made with othergermplasm, the map of the integration region can be compared to similarmaps for suspect plants, to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below.

1. Genes That Confer Resistance to Pests or Disease and That Encode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection (Rockville,Md.), for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

(C) A lectin. See, for example, the disclosure by Van Damme et al.,Plant Molec. Biol. 24: 25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

(D) A vitamin-binding protein, such as avidin. See PCT applicationUS93/06487 the contents of which are hereby incorporated by. Theapplication teaches the use of avidin and avidin homologues aslarvicides against insect pests.

(E) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793(1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huubet al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., Biosci.Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomycesnitrosporeus α-amylase inhibitor).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269: 9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm.163: 1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

(H) An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116: 165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

(I) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(K) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(L) A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

(M) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco raffle virus and tobacco mosaic virus. Id.

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(P) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10: 1436 (1992). The cloning and characterization of a gene whichencodes a bean endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2: 367 (1992).

(R) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to a Herbicide, For Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such As:

(A) Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89: 2624 (1992).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35: 383 (1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis α-amylase), Elliot et al., Plant Molec.Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes),Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102: 1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Maize Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton,1993) pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. See, for example, Kado, C. I., Crit. Rev.Plant Sci.10: 1 (1991). Descriptions of Agrobacterium vector systems andmethods for Agrobacterium-mediated gene transfer are provided by Gruberet al., supra, Miki et al., supra, and Moloney et al., Plant CellReports 8: 238 (1989). See also, U.S. Pat. No. 5,591,616, issued Jan. 7,1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and maize. Hieiet al., The Plant Journal 6: 271-282 (1994); U.S. Pat. No. 5,591,616,issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., Trends Biotech. 6: 299(1988), Klein et al., Bio/Technology 6: 559 563 (1988), Sanford, J. C.,Physiol Plant 79: 206 (1990), Klein et al., Biotechnology 10: 268(1992). In maize, several target tissues can be bombarded withDNA-coated microprojectiles in order to produce transgenic plants,including, for example, callus (Type I or Type II), immature embryos,and meristematic tissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4: 2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake ofDNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen.Genet.199: 161 (1985) and Draper et al., Plant Cell Physiol.23: 451(1982). Electroporation of protoplasts and whole cells and tissues havealso been described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al.,Plant Mol. Biol. 24: 51-61 (1994).

Following transformation of maize target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells,, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing transgenic inbred lines. Transgenic inbred lines could then becrossed, with another (non-transformed or transformed) inbred line, inorder to produce a transgenic hybrid maize plant. Alternatively, agenetic trait which has been engineered into a particular maize lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-elite lineinto an elite line, or from a hybrid maize plant containing a foreigngene in its genome into a line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

INDUSTRIAL APPLICABILITY

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry.Stalks and husks are made into paper and wallboard and cobs are used forfuel and to make charcoal.

The seed of the hybrid maize plant and various parts of the hybrid maizeplant and transgenic versions of the foregoing, can be utilized forhuman food, livestock feed, and as a raw material in industry.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationssuch as single gene modifications and mutations, somoclonal variants,variant individuals selected from large populations of the plants of theinstant hybrid may be practiced within the scope of the invention, aslimited only bit the scope of the appended claims.

DEPOSITS

Applicant has made a deposit of at least 2500 seeds of Hybrid Maize Line36D14 with the American Type Culture Collection (ATCC), Manassas, Va.20110 USA, ATCC Deposit No. PTA-1725. The seeds deposited with the ATCCon Apr. 18, 2000 were taken from the deposit maintained by PioneerHi-Bred International, Inc., 800 Capiatal Square, 400 Locust Street, DesMoines, Iowa 50309-2340 since prior to the filing date of thisapplication. This deposit of the Hybrid Maize Line 36D14, and itsparential lines, GE518397 and GE501400, will be maintained in the ATCCdepository, which is a public depository, for a period of 30 years, or 5years after the most recent request, or for the enforceable life of thepatent, whichever is longer, and will be replaced if it becomesnonviable during that period. Additionally, Applicant has satisfied allthe requirements of 37 C.F.R. §§1.801-1.809, including providing anindication of the viability of the samples. Applicant imposes norestrictions on the availability of the deposited material from theATCC; however, Applicant has no authority to wave any restrictionsimposed by law on the transfer of biological material or itstransportation in commerce. Applicant does not waive any infringement ofhis rights granted under this patent, or other patents and the PlantVariety Protection Act (7 USC 2321 et seq.) which may protect either theparental lines or Hybrid Maize Line 36D14.

What is claimed is:
 1. Hybrid maize seed designated 36D14,representative seed of said hybrid 36D14 having been deposited underATCC accession number PTA-1725.
 2. A maize plant, or its parts, producedby the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovuleof the plant of claim
 2. 5. A tissue culture of regenerable cells of ahybrid maize plant 36D14, representative seed of said hybrid maize plant36D14 having been deposited under ATCC, accession number PTA-1725,wherein the tissue regenerates plants capable of expressing all themorphological and physiological characteristics of said hybrid maizeplant 36D14.
 6. A tissue culture according to claim 5, the cells orprotoplasts being from a tissue selected from the group consisting ofleaves, pollen, embryos, roots, root tips, anthers, silks, flowers,kernels, ears, cobs, husks, and stalks.
 7. A maize plant, or its parts,regenerated from the tissue culture of claim 5 and capable of expressingall the morphological and physiological characteristics Of hybrid maizeplant 36D14, representative seed having been deposited under ATCCaccession number PTA-1725.
 8. A maize plant according to claim 2,wherein the genetic material of said plant contains one or moretransgenes.
 9. A maize plant according to claim 2, wherein the geneticmaterial of said plant contains one or more genes transferred bybackcrossing.
 10. A maize plant, or its parts wherein at least oneancestor of said maize plant is the maize plant, or its parts of claim9, said maize plant capable of expressing a combination of at least two36D14 traits selected from the group consisting of: a relative maturityof approximately 103 based on the Comparative Relative Maturity RatingSystem for harvest moisture of grain, consistent grain yield, aboveaverage late season plant health, above average stalk lodgingresistance, above average tolerance to Northern Corn Leaf Blight, aboveaverage tolerance to Stewart's Wilt, above average tolerance toAnthracnose Stalk Rot, above average brittle snap resistance, improvedresistance to Grav Leaf Spot, a substantial decree of imidazolinoneresistance, and is well suited to the Central Corn Belt and theNorthwest, North Central, Northeast, and Western regions of the UnitedStates and also to Canada, including Ontario.
 11. A maize plant, or itsparts, having all the morphological and physiological characteristics ofthe plant of claim
 2. 12. A maize plant, or its parts, wherein at leastone ancestor of said maize plant is the maize plant, or its parts, ofclaim 2, said maize plant capable of expressing a combination of traitsselected from the group consisting of: a relative maturity ofapproximately 103 based on the Comparative Relative Maturity RatingSystem for harvest moisture of grain, consistent grain yield, aboveaverage late season plant health, above average stalk lodgingresistance, above average tolerance to Northern Corn Leaf Blight, aboveaverage tolerance to Stewart's Wilt, above average tolerance toAnthracnose Stalk Rot, above average brittle snap resistance, improvedresistance to Gray Leaf Spot, a substantial degree of imidazolinoneresistance, and is well suited to the Central Corn Belt and theNorthwest, North Central, Northeast, and Western regions of the UnitedStates and also to Canada, including Ontario.
 13. A method fordeveloping a maize plant in a maize plant breeding program using plantbreeding techniques, which include employing a maize plant, or itsparts, as a source of plant breeding material, comprising: obtaining themaize plant, or its parts, of claim 2 as a source of said breedingmaterial.
 14. The maize plant breeding program of claim 13 wherein plantbreeding techniques are selected from the group consisting of: recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.
 15. A method for developing a maize plant in a maizeplant breeding program using plant breeding techniques, which includeemploying a maize plant, or its parts, as a source of plant breedingmaterial, comprising: obtaining the maize plant, or its parts, of claim8 as a source of said breeding material.
 16. The maize plant breedingprogram of claim 15 wherein plant breeding techniques are selected fromthe group consisting of: recurrent selection, backcrossing, pedigreebreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, and transformation.
 17. The maizeplant of claim 11 wherein said maize plant is male sterile.
 18. A methodfor developing a maize plant in a maize plant breeding program usingplant breeding techniques, which include employing a maize plant, or itsparts, as a source of plant breeding material, comprising: using themaize plant, or its parts, of claim 9 as a source of said breedingmaterial.
 19. The maize plant breeding program of claim 18 wherein plantbreeding techniques are selected from the group consisting of: recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.
 20. A maize plant, or its parts, wherein at least oneancestor of said maize plant is the maize plant, or its parts, of claim8, said maize plant capable of expressing a combination of at least two36D14 traits selected from the group consisting of: a relative maturityof approximately 103 based on the Comparative Relative Maturity RatingSystem for harvest moisture of grain, consistent grain yield, aboveaverage late season plant health, above average stalk lodgingresistance, above average tolerance to Northern Corn Leaf Blight, aboveaverage tolerance to Stewart's Wilt, above average tolerance toAnthracnose Stalk Rot, above average brittle snap resistance, improvedresistance to Gray Leaf Spot, a substantial degree of imidazolinoneresistance, and is well suited to the Central Corn Belt and theNorthwest, North Central, Northeast, and Western regions of the UnitedStates and also to Canada, including Ontario.
 21. A hybrid maize plantaccording to claim 11, wherein the genetic material of said plantcontains one or more transgenes.
 22. A maize plant, or its parts,wherein at least one ancestor of said maize plant is the maize plant, orits parts, of claim 21, said maize plant capable of expressing acombination of at least two 36D14 traits selected from the groupconsisting of: a relative maturity of approximately 103 based on theComparative Relative Maturity Rating System for harvest moisture ofgrain, consistent grain yield, above average late season plant health,above average stalk lodging resistance, above average tolerance toNorthern Corn Leaf Blight, above average tolerance to Stewart's Wilt,above average tolerance to Anthracnose Stalk Rot, above average brittlesnap resistance, improved resistance to Gray Leaf Spot, a substantialdegree of imidazolinone resistance, and is well suited to the CentralCorn Belt and the Northwest, North Central, Northeast, and Westernregions of the United States and also to Canada, including Ontario. 23.A method for developing a maize plant in a maize plant breeding programusing plant breeding techniques, which include employing a maize plant,or its parts, as a source of plant breeding material, comprising: usingthe maize plant, or its parts, of claim 21 as a source of said breedingmaterial.
 24. The maize plant breeding program of claim 23 wherein plantbreeding techniques are selected from the group consisting of: recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.
 25. A hybrid maize plant according to claim 11, whereinthe genetic material of said plant contains one or more genestransferred by backcrossing.
 26. A maize plant, or its parts, wherein atleast one ancestor of said maize plant is the maize plant, or its parts,of claim 25, said maize plant capable of expressing a combination of atleast two 36D14 traits selected from the group consisting of: a relativematurity of approximately 103 based on the Comparative Relative MaturityRating System for harvest moisture of grain, consistent grain yield,above average late season plant health, above average stalk lodgingresistance, above average tolerance to Northern Corn Leaf Blight, aboveaverage tolerance to Stewart's Wilt, above average tolerance toAnthracnose Stalk Rot, above average brittle snap resistance, improvedresistance to Gray Leaf Spot, a substantial degree of imidazolinoneresistance, and is well suited to the Central Com Belt and theNorthwest, North Central, Northeast, and Western regions of the UnitedStates and also to Canada, including Ontario.
 27. A method fordeveloping a maize plant in a maize plant breeding program using plantbreeding techniques, which include employing a maize plant, or itsparts, as a source of plant breeding material, comprising: using themaize plant, or its parts, of claim 25 as a source of said breedingmaterial.
 28. The maize plant breeding program of claim 27 wherein plantbreeding techniques are selected from the group consisting of: recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.
 29. A maize plant, or its parts, wherein at least oneancestor of said maize plant is the maize plant, or its parts, of claim11, said maize plant capable of expressing a combination of at least two36D14 traits selected from the group consisting of: a relative maturityof approximately 103 based on the Comparative Relative Maturity RatingSystem for harvest moisture of grain, consistent grain yield, aboveaverage late season plant health, above average stalk lodgingresistance, above average tolerance to Northern Corn Leaf Blight, aboveaverage tolerance to Stewart's Wilt, above average tolerance toAnthracnose Stalk Rot, above average brittle snap resistance, improvedresistance to Gray Leaf Spot, a substantial degree of imidazolinoneresistance, and is well suited to the Central Corn Belt and theNorthwest, North Central, Northeast, and Western regions of the UnitedStates and also to Canada, including Ontario.
 30. A method fordeveloping a maize plant in a maize plant breeding program using plantbreeding techniques, which include employing a maize plant, or itsparts, as a source of plant breeding material, comprising: using themaize plant, or its parts, of claim 11 as a source of said breedingmaterial.
 31. The maize plant breeding program of claim 30 wherein plantbreeding techniques are selected from the group consisting of: recurrentselection, backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.